Conifers in general, and pines in particular, are extremely important crop plants. This group of trees supplies fiber and wood to a large fraction of the world's population. Genetic improvement in pines has been limited by the long generation times of these plants, and the most advanced breeding programs have carried out no more than about three generations of selection. Genetic improvement by gene transfer methods has great potential application in this area, but methods for the introduction of foreign DNA and the subsequent production of transgenic plants, although available for many crop species, have been difficult to provide for the conifers.
Techniques have been developed for transforming conifers with Agrobacterium tumefaciens vectors. See, e.g., Sederoff, Stomp, Chilton, and Moore, Gene Transfer into Loblolly Pine by Agrobacterium tumefaciens, 4 Bio/Technology 647 (1986); see also U.S. Pat. No. 4,459,355 to Cello and Olsen. Such systems add complexity to methods for the genetic modification of plants because they add a second living system (the Agrobacterium species) to the method. More recently, in other plants, considerable interest has focused on the use of ballistic microprojectiles as DNA vectors for the transformation of plant cells. See, e.g., McCabe et al., Stable Transformation of Soybean (Glycine Max) by Particle Acceleration, 6 Bio/Technology 923 (1988); Klein et al., Transfer of Foreign Genes into Intact Maize Cells with High-Velocity Microprojectiles. 85 Proc. Natl. Acad. Sci. USA 4305 (1988). While these ballistic transformation methods appear attractive, it has not previously been suggested how they might be applied to the transformation of conifers.
The following problems in developing genetic engineering procedures for plants have been noted: First, unlike unicellular microorganisms, plant cells have a low rate of proliferation. Second, plant cells are much more sensitive to their environment in relation to viability, proliferation and regeneration to plants. Third, in order to establish whether a foreign gene has been usefully integrated into a plant cell, it is necessary to establish that the regenerated plant expresses the phenotype. Fourth, plant cells have rigid cell walls. See U.S. Pat. No. 4,795,855 to Fillatti and Comai, at Col. 1. In addition, when plant tissue is wounded during a transformation process, it can be difficult to obtain subsequent morphogenesis from the wounded, transformed tissue. Untransformed tissue within the treated tissue sample may undergo morphogenesis, but this is not the object of the transformation process.
Accordingly, an object of the present invention is to provide a method for transforming conifers with microprojectiles.
Another object of the present invention is to provide a method of transforming plant tissue in which the tissue transformed is capable of subsequently undergoing morphogenesis.
Another object of the present invention is to provide a method of transforming conifers which yields stable transformation.